The solid-phase synthesis of a peptide involves stepwise additions of amino-terminus-blocked amino acids to a peptide chain, the carboxyl terminus of which is anchored to a solid support. The synthesis begins with the amino acid at the carboxyl-terminal of the chain and proceeds with single- amino acid additions to the successive amino termini of the chain. It is initiated by covalently attaching the carboxyl terminus of the carboxyl-terminal amino acid to an insoluble solid support, which is typically a matrix of resin beads that are large enough to be separated from the liquid phase by filtration.
The next amino acid to be added is first protected at its amino terminus with a blocking group, such as butyloxycarbonyl (BOC), so that this terminus is no longer reactive with the reagents that promote the formation of peptide bonds. The blocked amino acid is then reacted, in the presence of a condensing agent, such as dicyclohexyl-carbodiimide, with the anchored amino acid to form a peptide bond between the carboxyl terminus of the bocked amino acid and the amino terminus of the anchored amino acid.
The resulting peptide chain (which now consists of two amino acids) remains anchored to the insoluble resin, and is therefore easily separated from the reactants by thorough washing. The blocking group is then removed from the amino terminus of the amino terminal amino acid of the peptide chain by acidification (using trifluoroacetic acid, for example,) so that the peptide is now terminated with a free amino group that is ready to react with the next blocked amino acid to be added to the chain.
In similar fashion, subsequent amino acids are added to the anchored peptide chain to build the complete peptide. After the peptide is completed, it is removed from the insoluble resin and isolated.
In reality, peptide synthesis is more complicated. For example, the reagents used to form the peptide bond also react with side groups of some of the amino acids (approximately twenty different amino acids are routinely used to form a peptide). Therefore, these sensitive side groups must also be protected with special blocking groups during the entire synthesis of the peptide. These special blocking groups must be stable under the conditions of deblocking of the amino-terminal termini of the peptide chains and must also be readily removed from the completed peptide.
Also, each amino acid has its own individual optimum reaction kinetics, and these kinetics are affected by the environment surrounding the amino terminal of the peptide chain. This environment is determined not only by the amino acid at the amino terminus itself, but also by the other amino acids in the peptide chain and their interactions with both the solid and the liquid phases.
Specifically, the different amino acid configurations may cause the peptide to bend and fold three-dimensionally, and thus the free amino terminus of the amino terminal amino acid may be hindered sterically from reacting with the blocked amino acid to be added to the peptide chain. Also, the different amino acids in the chain have different hydrophobic/hydrophilic effects on the surroundings of the free amino terminal, particularly, on the solvation of the resin itself, which affect the accessibility of the free amino terminus to the liquid reactants and, thus, the reaction rate. Therefore, since the sequence of each peptide to be synthesized is different, the optimum reaction conditions for each amino acid addition are difficult to predict.
As a result, the reaction at each step of the synthesis seldom goes to completion, that is, the yield is generally somewhat less than 100%. Obviously, the yield at each step must be very high if a peptide chain of substantial length is to be prepared in substantial quantity. For example, a yield of 99.0% per step results in a product yield of only 81% after 20 amino acid additions, while a yield per step increase of only 0.5% (to 99.5% per step) results in a product of 90% yield--almost 10% higher. Another increase of only 0.4% per step--to 99.9% results in a 98% ultimate yield. The yield represents not only the total amount of the resulting peptide, but also its purity. The purity is important because it is difficult and costly to separate the desired peptide from undesidred peptides that vary in only a few amino acids, and which arise from incomplete reactions. The total amount is important because if the yield is low, the amount of expensive starting materials must be increased accordingly.
A system for synthesizing these peptides must therefore accomodate the large number of different steps and the varying reaction conditions. It also must be constructed to minimize cross-contamination among the amino acids, as well as the solvents and reagents used in the process. Ideally, the system should further include a method and apparatus for monitoring the completeness of each amino acid addition before the next amino acid is added to the peptide chain. If the results from the monitoring indicate that the amino acid addition has not proceeded to a desired level of completeness, the previous addition reaction may be repeated until the desired level is attained. Such a system ensures the highest possible yield at each step.
Prior apparatus for synthesizing peptides can be divided into two types: column synthesizers, such as described in U.S. Pat. No. 4,362,699, and shaker/reactor vessels, which are described in U.S. Pat. Nos. 4,362,699, 3,531,258, 3,647,390, and 3,557,077. Prior monitoring methods and the advantaegs and disadvantages of each method are summarized in "Selected Methods for Monitoring a Solid-Phase Peptide Synthesis", G. Barany and B. Merrifield, The Peptides, V.2., 1979, pp. 150-154.
In the column synthesizers, solid support beads to which the growing peptide chains are attached, are packed into a column. The reagents, solvents, and amino acids required for synthesizing the peptide are reacted with the solid support by passing them sequentially through the column. To obtain reasonable flow rates, these column synthesizers are operated under high pressure, usually greater than 200 psi. With unidirectional flow through the column, the high pressure may compress the solid supports, thus causing increased back pressure and pumping problems. These pressure problems require that special precautions be taken in the system design.
The prior shaker/reactor systems contain filters made from glass frits for retaining a particulate insoluble matrix in a reactor vessel while allowing passage of liquid and gas. Generally, these reactor vessels have separate inlets and outlets for unidirectional flow through the reactor and its filter, such as described in U.S. Pat. No. 3,521,258, to Merrifield et al., and in U.S. Pat. No. 3,557,077 to Brundfeldt et al.
During the various intermediate steps required for each amino acid addition, the different solvents cause the solid support to swell and shrink. The shrunken beads may enter the pores in the filter during one step, and swell during a subsequent step, thereby trapping the beads in the filter. Since flow through the filter is unidirectional, the trapped beads are not removed from the filter, and eventually, during the course of peptide synthesis, they may accumulate in sufficient quantity to impede flow through the filter. Also, clogging of the filter makes the trapped beads at least partially inaccessible to the reaction solvents and reagents, resulting in incomplete reactions at each step.
In the Merrifield Pat. No. 3,521,258, this clogging of the filter causes a backpressure buildup in the system that makes it difficult to obtain a closely metered flow into the reactor. Close metering is important because, with an increase in the degree of uncertainty in the transfer, a corresponding increase in the amount of expensive reagents which must be transferred to compensate for this uncertainty.
There are other drawbacks in these prior systems. For example, in the Merrifield Patent, crosscontamination between solvents and reagents in the selective valves and pumps is a problem. Also, this device cannot accomodate a wide range of reaction volumes because the metering pump is adjustable only over a relatively narrow volume range. Thus, it cannot be used to produce both analytical (small) and commercial (large) quantities of peptides.
Kubodera et al., U.S. Pat. No. 3,647,390, describes a system that avoids clogging of the filter. The reaction vessel has a single port for both inflow and outflow. There is a single filter between this port and a reaction chamber. Thus each time liquid ingredients or reagents are added to the vessel, their flow through the filter tends to dislodge matrix beads that were retained on the filter during the preceding removal of liquid from the vessel. In Kubodera et al., liquid from the reservoirs is transferred to an intermediate metering vessel, and subsequently, from the intermediate metering vessel to the reaction vessel. The metering is accomplished by drawing a vacuum on a vacuum chamber, and connecting the chamber to the intermediate metering vessel. The resulting pressure decrease in the intermediate metering vessel causes transfer of liquid from the reservoir to the intermediate metering vessel until the pressure in the intermediate metering vessel and the vacuum chamber increase to standard pressure. Thus the amount of liquid transferred is directly related to the volumes of the intermediate metering vessel and the vacuum chamber.
This system is cumbersome in that a significant and high vacuum must be drawn on the vacuum chamber for accurate metering of the various liquids. Also, it is difficut to vary the amount drawn to cope with different size reaction vessels, or different quantities or amounts. Essentially, one must change to different-size vacuum chambers. This system also presents a significant likelihood of cross-contamination in the intermediate metering vessel, because it is difficult to completely remove the various liquids from the walls of the intermediate metering vessel before subsequent liquids are introduced.
Of the various methods for monitoring peptide synthesis, for reasons of sensitivity, the ideal methods assay the solid phase for the unreacted, or "free" amino groups at the amino terminus of the anchored peptide chains. Furthermore, these ideal methods should not destroy the peptide chains or interfere with the synthesis of the peptides. The known methods that meet these requirements involve titration of the resin-bound amino groups with salt-forming reagents and quantitation of the amount of resin-bound reagent either potentiometrically or spectrophotometrically.
These titration methods utilize three different reagents: pyridine hydrochloride, perchloric acid or picric acid. In the pyridine hydrochloride method, free amino groups on the solid phase are converted to a hydrochloride salt with pyridine hydrochloride in methylene chloride. The chloride is subsequently eluted from the solid phase to the liquid phase, the liquid phase is separated from the solid phase and the level of chloride ion in the liquid phase is determined potentiometrically. In the perchloric acid method, the free amino groups on the solid phase are determined by direct potentiometric titration of the solid phase by insertion of an electrode directly into the reaction vessel. In the picric acid method, picrate salts of any free amino groups on the solid phase are formed with picric acid, a readily detected chromophore. The picrate ion is subsequently eluted from the solid phase to the liquid phase, the liquid phase is separated from the solid phase, and the level of the picrate ion in the liquid phase is determined spectrophotemetrically.
These titration methods have some disadvantages. The acids used in the pyridine hydrochloride titration can remove some of the amino-blocking groups. In the perchloric acid method, some of the acid sensitive groups may be lost and the amino groups can become permanently acetylated. In the picric acid method, the chromophore reacts stoichiometrically with the amino acid histidine, including its protected forms. Thus, although this increased reactivity can be accounted for by corresponding predictable increasess in the background, the increased background destroys the sensitivity of the assay for free amino ends when peptides containing histidine are monitored. This lack of sensitivity is alleviated to some extent by the use of immidazolium picrate, which reacts less strongly than picric acid with histidine residues.
Also, because the monitoring agents used in these titration methods involve ionic interactions, the agents interact to some extent with the other amino acids in the peptide chain. Therefore, as the peptide chain length increses, so do the number of interactions. The consequence is higher backgrounds, more false positives, and lower sensitivity.